International Journal of Environmental Science and Development, Vol. 4, No. 2, April 2013

239DOI: 10.7763/IJESD.2013.V4.343

Abstract—The production of bio-ethanol from corn is a

mature technology that is not likely to see significant reduction

in the production costs. Substantial cost reductions may be

possible if cellulose based agricultural wastes such as corn cobs

are used instead of corn. In this study, corn cobs which are in

abundance and do not interfere with food security was

subjected to simultaneous saccharification and fermentation

process by co-culture of Aspergillus niger and Saccharomyces

cerevisiae for 7 days. The corn cobs were sundried, milled into

powder using hammer milling and stored at room temperature

(25O

C) before use. The growth media used for culturing

Aspergilus niger and Saccharomyces cerevisiae inocula were

prepared respectively. Parameters such as biomass yield, cell

dry weight, reducing sugar concentration, pH of the

fermentation medium and the ethanol yield were determined at

24 hours intervals. The results of the study revealed that the

yeast and mould biomass yield obtained from the corn cobs on

the 7th

day was 0.59 (OD), while the microbial cell dry weight

obtained on the same day was 0.88mg/cm3. The substrate was

hydrolyzed to produce 0.63mg/cm3 reducing sugar

concentrations. The pH values of the fermentation medium

varied between 3.05 and 7.58. Optimal ethanol yield of 10.08v/v

was obtained after 7 days of fermentation. The results of this

study suggest that agricultural wastes that contain fermentable

sugars can no longer be discarded into our environment, but

should be converted to useful products like bio-ethanol.

Index Terms—Saccharification, fermentation, corn cobs,

bioethanol.

I. INTRODUCTION

Lignocelllosic biomass can be utilized to produce ethanol,

a promising alternative energy for the limited crude oil [1].

There are mainly two process involved in the conversion

hydrolysis of cellulose in the lignocellulosic biomass to

produce reducing sugars and fermentation of the sugars to

ethanol [2]-[5]. The hydrolysis of cellulose is usually

catalysed by cellulase enzymes, and fermentation is carried

by yeasts or bacteria. During the enzymatic hydrolysis,

cellulose is degraded by the cellulase to reducing sugars that

can be fermented by yeast or bacteria to ethanol [1] the

optimization of cellulose enzymes and enzymes loading can

also improve the hydrolysis. Simultaneous saccharification

and fermentation effectively removes glucose, which is an

Manuscript received December 5, 2012; revised February 5, 2013.

J. Itelima and A. Ogbonna are with the Department of Plant Science and

Technology.

S. Pandukur is with the Department of Science Laboratory Technology.

J. Egbere is with the Department of Microbiology all of University of Jos

Nigeria.

A. Salami is with the University of Jos, Nigeria (e-mail:

janetitelima@yahoo.com).

inhibitory to cellulose activity, thus increasing the yield and

rate of cellulose hydrolysis. The contents of cellulose,

hemicellulose and lignin in corn cob are 45%, 35% and 15%

respectively [3], [6]. Lignocellulosic feed stocks such as

agricultural wastes have favourable utilization potential for

bio-ethanol production because of their quantity and

competitive price. The main contributive parameter of

bio-ethanol is the cost of the raw material and in order to

reduce the overall cost of production corn cob which is

abundant and do not interfere with food security was used for

this experiment. Apart from the solvent nature of ethanol, it

could also serve as a basic raw material for the synthesis of

other products. It is also a safer alternative to methyl tertiary

butyl ether (MTBE), which is usually added to gasoline in

order to achieve a better and healthier combustion [7]. The

United States Environmental Protection Agency announced

its intentions to regulate MTBE addition to gasoline because

of its toxic nature and its possible role in the contamination of

community water sources [8]. In view of this, the demand for

ethanol could further increase [9]. The objective of this study

was therefore to produce ethanol from a cheap commonly

available agricultural waste such as corn cob with aid of a

co-culture of Aspergillus niger and Saccharomyces

cerevisiae.

II. MATERIALS AND METHODS

A. Preparation of Corn Cobs for Hydrolysis

A total of 40 corn cobs weighing 70-120g each were dried

in the sun and then pulverized with the aid of a hammer mill.

The resultant corn powder was sieved with a fine- mesh sieve.

A weight of 45g of the resultant maize cob powder was then

suspended in 500ml distilled water in 1000ml conical flask.

The flask with its contents was then autoclaved at 121OC at

151b pressure for a period of 15 minutes after which it was

cooled to room temperature.

B. Preparation of Growth Medium

A. niger growth medium was prepared by mixing D-

mannitol (50.0g), NaN02 (2.0g), K2HP04 (0.35g),

MgS04 .7H20 (0.25g), FeS04.7H20 (0.001g), ZnS04

(0.00088g), CuS04. 5H20 (0.0002g), MnS04. 4H20 (0.00012g),

NaMo04. 2H20 (0.00005g), Distilled water (1000ml). The pH

was adjusted to 4 with H2S04 and the whole mixture was then

sterilized by autoclaving at 1210C at 15psi for 15minutes.

Main while, Saccharomyces cerevisiae growth medium was

prepared using yeast – malt broth at pH 5.5 [10].

C. Preparation of Inocula and Fermentation Procedure

A. niger inoculum was prepared in 250cm3

Simultaneous Saccharification and Fermentation of Corn

Cobs to Bio-Ethanol by Co-Culture of Aspergillus Niger

and Saccharomyces Cerevisiae

J. Itelima, A. Ogbonna, S. Pandukur, J. Egbere, and A. Salami

International Journal of Environmental Science and Development, Vol. 4, No. 2, April 2013

240

cotton–plugged conical flask containing 100cm3 of the

different substrates growth media. The flasks were sterilized

and inoculated with 0.11 (OD) A. niger spores. Each of the

flasks was incubated on a shaker with agitation rate of 300

rpm at 300C for five days. S. cerevisiae inoculum was

prepared in the same way as the A. niger inoculum except that

yeast malt broth was used. The growth medium was

inoculated with 0.08 (OD) yeast cells and incubated for 24

hours. The fermentation medium used for ethanol production

was identical to the growth medium as indicated above.

Ethanol fermentation was carried out in 500cm3 conical

flasks each containing 300cm3 of medium. The medium was

sterilized and inoculated with 5% (v/v) growth media

containing A. niger and S. cerevisiae and incubated on a

shaker with an agitation rate of 300rpm at 300C for seven

days.

D. Analytical Procedure

Thirty cubic centimeters (30cm3) of the sample was

collected from the flask at 24 hours interval and 27cm3 was

centrifuged at 400rpm for 30 minutes to remove the cells. The

supernatant fluid was filtered through whatman filter paper

No.1 and the filtrate was used for determining ethanol and

reducing sugar concentration. The remaining 3cm3 was used

to determine the cell density.

E. Determination of Cell Density

Three cubic centimeters (3cm3) of the sample was used to

determine the cell density at 690nm using CECILCE 1020

spectrophotometer. The spectrophotometer was blanked with

an uninoculated fermentation medium.

F. Determination of Cell Dry Weight

The residue (cells) obtained after centrifugation was

filtered using whatman filter paper No.1, washed with

distilled water to remove the residual substrates and dried in

the hot air oven at 700C. The filter paper was pre–weighed

before filtering and reweighing after drying until a constant

weighed was obtained. Thus, the cell dry weights were

determined as follows: Cell dry weight = weight of filter

paper and cell after drying – weight of filter paper only.

G. Qualitative and Quantitative Analysis of Reducing

Sugar Present in the Samples

The qualitative analysis was carried out using Benedict’s

solution (Amadi et al., 2004), while the quantitative analysis

was carried out using 3, 5–dinitrosalicylic acid. The

concentration of the reducing sugar present in the samples

was determined by adding 1cm3 of 3, 5 – dinitrosalicylic acid

to 1cm3 of each of the samples and boiled for 5 minutes and

10cm3 distilled water was added. The absorbance of each of

sample was determined at 540nm using JENWAY 6400

spectrophotometer. Thus, the concentration values were

extrapolated from the glucose standard curve [11].

H. Qualitative and Quantitative Analysis of Ethanol

Present in the Distillate

The filtrates were distilled at 700C using rotary evaporator.

The qualitative analysis was carried out using ethanolic acid.

Two cubic centimeters of ethanolic acid was added to 1cm3

of the distillate and heated in the water bath for 5 minutes

until characteristics sweet smell of esters was perceived.

Thus, concentrations of the distillates were extrapolated

from the density standard curve. Furthermore, the pH,

refractive index, and specific gravity values were

extrapolated from the standard curves prepared from each

parameter since the concentrations of the distillates were

known [11].

III. RESULTS AND DISCUSSION

A. Biomass Yield and Cell Dry Weight

The yeast and mould (biomass) yield was obtained by

determining the absorbance of the samples at 690nm which

represented the cell density (Fig. 1). The yeast and mould

biomass yield obtained from corn cobs (CC) fermentation

medium increased gradually from 0.03 (OD) on the first day

to 0.59 (OD) on the seventh day. The microbial cell dry

weights obtained increased from 0.47mg/cm3 on the first day

to 0.88mg/cm3 on the seventh day respectively (Fig. 2).

Fig. 1. Yeast and mould yield from corn cobs fermentation medium

Fig. 2. Microbial cell dry weight obtained from fermentation medium

B. Reducing Sugar Concentration

The ability of the Aspergillus niger amylase and cellulase

to breakdown the corn cobs into reducing sugar was studied.

The results are represented in Fig. 3 in terms of the amount of

reducing sugars (mg/cm3) produced at 24 hours interval for

seven days. Corn cob was rapidly hydrolyzed to produce 0.63

mg/cm3 reducing sugar and the concentration decreased

gradually as the fermentation period increased.

C. The pH Values of the Fermentation Medium

The results of the pH values of the corn cob fermentation

medium are shown in (Fig. 4). The values ranged between

3.05 and 7.58. The gradual decrease in pH as recorded in this

study is an indication that acids are accumulated in the

fermentation medium during the fermentation process. This

International Journal of Environmental Science and Development, Vol. 4, No. 2, April 2013

241

agrees with the report of [2] that A. niger produces organic and by fermentation.

D. Ethanol Field Obtained from Corn Cobs Fermentation The results of the ethanol yield are shown in Fig. 5.

Ethanol yield of 1.87% (v/v) was obtained on the first day and gradually increased to 10.08% (v/v). This result is not comparable to that obtained from ethanol produced from ground corn [12]. Reference [12] showed that 2.69kg of ground corn yielded 95% pure ethanol. Although ground corn gave a higher ethanol yield than corn cobs, the cost of production from corn is one of the most significant factors affecting the economy of ethanol; hence efforts are more concentrated on using cheap raw materials such as agricultural wastes.

Fig. 3. Reducing Sugar Concentration Obtained from Corn cobs

Fig. 4: pH of Corn Cobs Fermentation Medium

Fig. 5. Ethanol Yield obtained from Corn Cobs

The findings of this study have provided practical examples and gave a broad overview of the current status of

ethanol fermentation including biomass resources, microorganisms and technology. Also, the promising prospects of ethanol fermentation are especially highlighted. The significant of the study included are fermentation technology converting agricultural wastes (corn cobs) to ethanol, cellulase enzyme from A. niger utilized in the hydrolysis of lignocelluloses materials, immobilization of the microorganism in large quantity, simultaneous and fermentation and sugar conversion into ethanol.

Fuel ethanol production from plant biomass is of great economic and environmental significance [13]. The gradual increase in cell densities suggested that substantially amount of carbon was utilized for ethanol production instead of cell production and this is due to the ability of the yeast S. cerevisiae to ferment the sugar to ethanol. The susceptibility of sugars obtained after hydrolysis of corn cobs by A. niger to the fermentation activity of S. cerevisiae is significantly dependent on the composition of the sugar (Fig. 3). This is because various mixtures of the hexoses (e.g. glucose and mannose) and pentoses (e.g. xylose and arabinose) are released from the hydrolysis of lignocellulosic materials [14]. The fermentation process is significantly dependent on the effectiveness of sugar transporters of S. cerevisiae cells at translocating different sugars across the cell membrane. Sugar transporters are membrane bound proteins that take up sugar from the environment and deliver them to the metabolic pathways inside the cell [13]. The result of the present study agrees with the report of [10] that stated that most substrates are utilized for ethanol production in co-culture fermentation (Fig. 5). This study has demonstrated that agricultural lignocellulosic wastes such corn cobs are potential source for the production of bio-ethanol by enzymatic and microbial methods.

The results of the study clearly showed that simultaneous saccharification and fermentation of corn cobs to ethanol by a mixture of starch digesting fungus A. niger and a non-starch digesting sugar fermenter S. cerevisiae is feasible. The results of this study suggest that agricultural wastes that contain fermentable sugars can no longer be discarded into our environment, but should be converted to useful products like bio-ethanol. Based on this study the following recommendations are made; Biomass availability is the primary factor. A favourable region for bio – ethanol industrial production should have surplus biomass and no problem with food security. Economic factors such as land availability, labour, taxation, utilities, crop processing costs and transportation must be put into consideration otherwise there will be no market for it or no profit for its production even though it is production from renewable resource.

ACKNOWLEDGMENT

We wish to thank the Department of Plant Science and Technology, Department of Chemistry and the Department of Biochemistry all of University of Jos, Nigeria for providing the chemicals and materials we used during the course of this work.

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J. Itelima was born on 31, Januray, 1962 in Plateau

State, Nigeria. She went to BSc (HONS) Botany in

1988, University of Jos, Nigeria. She applied her MSc

Microbiology and Plant Pathology in 1993 in the

University of Jos, Nigeria. Her major field of study is

applied microbiology and plant pathology. Her

current research interest include Bio-fuel, Bio-

Fertilizer and Infectious microorganisms, Previous

research interest: Research on Medicinal plants Current Job location:

Department of Plant Science and Technology, Faculty of Natural Science,

University of Jos, Nigeria.